RESUMO
The dynamics of CD4+ T cell memory development remain to be examined at genome scale. In malaria-endemic regions, antimalarial chemoprevention protects long after its cessation and associates with effects on CD4+ T cells. We applied single-cell RNA sequencing and computational modelling to track memory development during Plasmodium infection and treatment. In the absence of central memory precursors, two trajectories developed as T helper 1 (TH1) and follicular helper T (TFH) transcriptomes contracted and partially coalesced over three weeks. Progeny of single clones populated TH1 and TFH trajectories, and fate-mapping suggested that there was minimal lineage plasticity. Relationships between TFH and central memory were revealed, with antimalarials modulating these responses and boosting TH1 recall. Finally, single-cell epigenomics confirmed that heterogeneity among effectors was partially reset in memory. Thus, the effector-to-memory transition in CD4+ T cells is gradual during malaria and is modulated by antiparasitic drugs. Graphical user interfaces are presented for examining gene-expression dynamics and gene-gene correlations ( http://haquelab.mdhs.unimelb.edu.au/cd4_memory/ ).
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Memória Imunológica , Malária/imunologia , Plasmodium/imunologia , Transcriptoma , Transferência Adotiva , Animais , Antimaláricos/farmacologia , Biomarcadores , Cromatina/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Malária/parasitologia , Malária/terapia , Camundongos , Plasmodium/efeitos dos fármacosRESUMO
Immune-modulating therapies have revolutionized the treatment of chronic diseases, particularly cancer. However, their success is restricted and there is a need to identify new therapeutic targets. Here, we show that natural killer cell granule protein 7 (NKG7) is a regulator of lymphocyte granule exocytosis and downstream inflammation in a broad range of diseases. NKG7 expressed by CD4+ and CD8+ T cells played key roles in promoting inflammation during visceral leishmaniasis and malaria-two important parasitic diseases. Additionally, NKG7 expressed by natural killer cells was critical for controlling cancer initiation, growth and metastasis. NKG7 function in natural killer and CD8+ T cells was linked with their ability to regulate the translocation of CD107a to the cell surface and kill cellular targets, while NKG7 also had a major impact on CD4+ T cell activation following infection. Thus, we report a novel therapeutic target expressed on a range of immune cells with functions in different immune responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Inflamação/imunologia , Células Matadoras Naturais/imunologia , Leishmania donovani/fisiologia , Leishmaniose Visceral/imunologia , Malária/imunologia , Proteínas de Membrana/metabolismo , Plasmodium/fisiologia , Animais , Células Cultivadas , Citotoxicidade Imunológica , Modelos Animais de Doenças , Exocitose , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Vesículas Secretórias/metabolismoRESUMO
Plasmodium parasites invade and multiply inside red blood cells (RBC). Through a cycle of maturation, asexual replication, rupture and release of multiple infective merozoites, parasitised RBC (pRBC) can reach very high numbers in vivo, a process that correlates with disease severity in humans and experimental animals. Thus, controlling pRBC numbers can prevent or ameliorate malaria. In endemic regions, circulating parasite-specific antibodies associate with immunity to high parasitemia. Although in vitro assays reveal that protective antibodies could control pRBC via multiple mechanisms, in vivo assessment of antibody function remains challenging. Here, we employed two mouse models of antibody-mediated immunity to malaria, P. yoelii 17XNL and P. chabaudi chabaudi AS infection, to study infection-induced, parasite-specific antibody function in vivo. By tracking a single generation of pRBC, we tested the hypothesis that parasite-specific antibodies accelerate pRBC clearance. Though strongly protective against homologous re-challenge, parasite-specific IgG did not alter the rate of pRBC clearance, even in the presence of ongoing, systemic inflammation. Instead, antibodies prevented parasites progressing from one generation of RBC to the next. In vivo depletion studies using clodronate liposomes or cobra venom factor, suggested that optimal antibody function required splenic macrophages and dendritic cells, but not complement C3/C5-mediated killing. Finally, parasite-specific IgG bound poorly to the surface of pRBC, yet strongly to structures likely exposed by the rupture of mature schizonts. Thus, in our models of humoral immunity to malaria, infection-induced antibodies did not accelerate pRBC clearance, and instead co-operated with splenic phagocytes to block subsequent generations of pRBC.
Assuntos
Malária/imunologia , Malária/metabolismo , Plasmodium/crescimento & desenvolvimento , Animais , Anticorpos Antiprotozoários/metabolismo , Modelos Animais de Doenças , Eritrócitos/microbiologia , Eritrócitos/fisiologia , Humanos , Camundongos , Parasitos , Fagócitos , Plasmodium/metabolismo , Plasmodium/patogenicidade , Plasmodium chabaudi/imunologia , Plasmodium chabaudi/patogenicidade , Plasmodium yoelii/imunologia , Plasmodium yoelii/patogenicidadeRESUMO
CD4+ Th cell differentiation is crucial for protecting against blood-stage Plasmodium parasites, the causative agents of malaria. It has been known for decades that more than one type of Th cell develops during this infection, with early models proposing a biphasic Th1/Th2 model of differentiation. Over the past decade, a large body of research, in particular, reports over the past 2-3 y, have revealed substantial complexity in the Th differentiation program during Plasmodium infection. In this article, we review how several studies employing mouse models of malaria, and recent human studies, have redefined the process of Th differentiation, with a particular focus on Th1 and T follicular helper (Tfh) cells. We review the molecular mechanisms that have been reported to modulate Th1/Tfh differentiation, and propose a model of Th1/Tfh differentiation that accommodates observations from all recent murine and human studies.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Malária/imunologia , Células Th1/imunologia , Animais , Humanos , Ativação Linfocitária/imunologia , Plasmodium/imunologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Differentiation of CD4+ Th cells is critical for immunity to malaria. Several innate immune signaling pathways have been implicated in the detection of blood-stage Plasmodium parasites, yet their influence over Th cell immunity remains unclear. In this study, we used Plasmodium-reactive TCR transgenic CD4+ T cells, termed PbTII cells, during nonlethal P. chabaudi chabaudi AS and P. yoelii 17XNL infection in mice, to examine Th cell development in vivo. We found no role for caspase1/11, stimulator of IFN genes, or mitochondrial antiviral-signaling protein, and only modest roles for MyD88 and TRIF-dependent signaling in controlling PbTII cell expansion. In contrast, IFN regulatory factor 3 (IRF3) was important for supporting PbTII expansion, promoting Th1 over T follicular helper (Tfh) differentiation, and controlling parasites during the first week of infection. IRF3 was not required for early priming by conventional dendritic cells, but was essential for promoting CXCL9 and MHC class II expression by inflammatory monocytes that supported PbTII responses in the spleen. Thereafter, IRF3-deficiency boosted Tfh responses, germinal center B cell and memory B cell development, parasite-specific Ab production, and resolution of infection. We also noted a B cell-intrinsic role for IRF3 in regulating humoral immune responses. Thus, we revealed roles for IRF3 in balancing Th1- and Tfh-dependent immunity during nonlethal infection with blood-stage Plasmodium parasites.
Assuntos
Diferenciação Celular/imunologia , Fator Regulador 3 de Interferon/imunologia , Malária/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Células Th1/imunologia , Animais , Feminino , Centro Germinativo/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Baço/imunologiaRESUMO
Severe malaria and associated high parasite burdens occur more frequently in humans lacking robust adaptive immunity to Plasmodium falciparum Nevertheless, the host may partly control blood-stage parasite numbers while adaptive immunity is gradually established. Parasite control has typically been attributed to enhanced removal of parasites by the host, although in vivo quantification of this phenomenon remains challenging. We used a unique in vivo approach to determine the fate of a single cohort of semisynchronous, Plasmodium berghei ANKA- or Plasmodium yoelii 17XNL-parasitized red blood cells (pRBCs) after transfusion into naive or acutely infected mice. As previously shown, acutely infected mice, with ongoing splenic and systemic inflammatory responses, controlled parasite population growth more effectively than naive controls. Surprisingly, however, this was not associated with accelerated removal of pRBCs from circulation. Instead, transfused pRBCs remained in circulation longer in acutely infected mice. Flow cytometric assessment and mathematical modeling of intraerythrocytic parasite development revealed an unexpected and substantial slowing of parasite maturation in acutely infected mice, extending the life cycle from 24 h to 40 h. Importantly, impaired parasite maturation was the major contributor to control of parasite growth in acutely infected mice. Moreover, by performing the same experiments in rag1-/- mice, which lack T and B cells and mount weak inflammatory responses, we revealed that impaired parasite maturation is largely dependent upon the host response to infection. Thus, impairment of parasite maturation represents a host-mediated, immune system-dependent mechanism for limiting parasite population growth during the early stages of an acute blood-stage Plasmodium infection.
Assuntos
Interações Hospedeiro-Parasita , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Plasmodium berghei/fisiologia , Plasmodium falciparum/fisiologia , Imunidade Adaptativa , Animais , Citocinas/metabolismo , Eritrócitos/parasitologia , Feminino , Citometria de Fluxo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/genética , Sistema Imunitário , Inflamação , Malária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Teóricos , Plasmodium yoelii/fisiologiaRESUMO
We describe an MHC class II (I-Ab)-restricted TCR transgenic mouse line that produces CD4+ T cells specific for Plasmodium species. This line, termed PbT-II, was derived from a CD4+ T cell hybridoma generated to blood-stage Plasmodium berghei ANKA (PbA). PbT-II cells responded to all Plasmodium species and stages tested so far, including rodent (PbA, P. berghei NK65, Plasmodium chabaudi AS, and Plasmodium yoelii 17XNL) and human (Plasmodium falciparum) blood-stage parasites as well as irradiated PbA sporozoites. PbT-II cells can provide help for generation of Ab to P. chabaudi infection and can control this otherwise lethal infection in CD40L-deficient mice. PbT-II cells can also provide help for development of CD8+ T cell-mediated experimental cerebral malaria (ECM) during PbA infection. Using PbT-II CD4+ T cells and the previously described PbT-I CD8+ T cells, we determined the dendritic cell (DC) subsets responsible for immunity to PbA blood-stage infection. CD8+ DC (a subset of XCR1+ DC) were the major APC responsible for activation of both T cell subsets, although other DC also contributed to CD4+ T cell responses. Depletion of CD8+ DC at the beginning of infection prevented ECM development and impaired both Th1 and follicular Th cell responses; in contrast, late depletion did not affect ECM. This study describes a novel and versatile tool for examining CD4+ T cell immunity during malaria and provides evidence that CD4+ T cell help, acting via CD40L signaling, can promote immunity or pathology to blood-stage malaria largely through Ag presentation by CD8+ DC.
Assuntos
Apresentação de Antígeno , Linfócitos T CD4-Positivos/imunologia , Antígenos CD40/imunologia , Células Dendríticas/imunologia , Malária/imunologia , Camundongos Transgênicos/imunologia , Parasitemia/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos de Protozoários/imunologia , Antígenos CD40/deficiência , Ligante de CD40/imunologia , Células Cultivadas , Cruzamentos Genéticos , Hibridomas , Ativação Linfocitária , Malária Cerebral/imunologia , Malária Cerebral/prevenção & controle , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/genética , Plasmodium berghei/imunologia , Quimera por RadiaçãoRESUMO
Parasite-specific antibodies protect against blood-stage Plasmodium infection. However, in malaria-endemic regions, it takes many months for naturally-exposed individuals to develop robust humoral immunity. Explanations for this have focused on antigenic variation by Plasmodium, but have considered less whether host production of parasite-specific antibody is sub-optimal. In particular, it is unclear whether host immune factors might limit antibody responses. Here, we explored the effect of Type I Interferon signalling via IFNAR1 on CD4+ T-cell and B-cell responses in two non-lethal murine models of malaria, P. chabaudi chabaudi AS (PcAS) and P. yoelii 17XNL (Py17XNL) infection. Firstly, we demonstrated that CD4+ T-cells and ICOS-signalling were crucial for generating germinal centre (GC) B-cells, plasmablasts and parasite-specific antibodies, and likewise that T follicular helper (Tfh) cell responses relied on B cells. Next, we found that IFNAR1-signalling impeded the resolution of non-lethal blood-stage infection, which was associated with impaired production of parasite-specific IgM and several IgG sub-classes. Consistent with this, GC B-cell formation, Ig-class switching, plasmablast and Tfh differentiation were all impaired by IFNAR1-signalling. IFNAR1-signalling proceeded via conventional dendritic cells, and acted early by limiting activation, proliferation and ICOS expression by CD4+ T-cells, by restricting the localization of activated CD4+ T-cells adjacent to and within B-cell areas of the spleen, and by simultaneously suppressing Th1 and Tfh responses. Finally, IFNAR1-deficiency accelerated humoral immune responses and parasite control by boosting ICOS-signalling. Thus, we provide evidence of a host innate cytokine response that impedes the onset of humoral immunity during experimental malaria.
Assuntos
Anticorpos Antiprotozoários/imunologia , Imunidade Humoral/imunologia , Proteína Coestimuladora de Linfócitos T Induzíveis/imunologia , Malária/imunologia , Receptor de Interferon alfa e beta/imunologia , Animais , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Plasmodium chabaudi/imunologia , Plasmodium yoelii/imunologia , Transdução de Sinais/imunologiaRESUMO
Naive CD4+ T cells must differentiate in order to orchestrate immunity to Plasmodium, yet understanding of their emerging phenotypes, clonality, spatial distributions, and cellular interactions remains incomplete. Here, we observe that splenic polyclonal CD4+ T cells differentiate toward T helper 1 (Th1) and T follicular helper (Tfh)-like states and exhibit rarer phenotypes not elicited among T cell receptor (TCR) transgenic counterparts. TCR clones present at higher frequencies exhibit Th1 skewing, suggesting that variation in major histocompatibility complex class II (MHC-II) interaction influences proliferation and Th1 differentiation. To characterize CD4+ T cell interactions, we map splenic microarchitecture, cellular locations, and molecular interactions using spatial transcriptomics at near single-cell resolution. Tfh-like cells co-locate with stromal cells in B cell follicles, while Th1 cells in red pulp co-locate with activated monocytes expressing multiple chemokines and MHC-II. Spatial mapping of individual transcriptomes suggests that proximity to chemokine-expressing monocytes correlates with stronger effector phenotypes in Th1 cells. Finally, CRISPR-Cas9 gene disruption reveals a role for CCR5 in promoting clonal expansion and Th1 differentiation. A database of cellular locations and interactions is presented: https://haquelab.mdhs.unimelb.edu.au/spatial_gui/.
Assuntos
Linfócitos T CD4-Positivos , Diferenciação Celular , Malária , Animais , Camundongos , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Malária/imunologia , Malária/parasitologia , Camundongos Endogâmicos C57BL , Fenótipo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores CCR5/metabolismo , Receptores CCR5/genética , Baço/imunologia , Células Th1/imunologiaRESUMO
Children in malaria-endemic regions can experience repeated Plasmodium infections over short periods of time. Effects of re-infection on multiple co-existing CD4+ T cell subsets remain unresolved. Here, we examine antigen-experienced CD4+ T cells during re-infection in mice, using scRNA-seq/TCR-seq and spatial transcriptomics. TCR transgenic TEM cells initiate rapid Th1/Tr1 recall responses prior to proliferating, while GC Tfh counterparts are refractory, with TCM/Tfh-like cells exhibiting modest non-proliferative responses. Th1-recall is a partial facsimile of primary Th1-responses, with no upregulated effector-associated genes being unique to recall. Polyclonal, TCR-diverse, CD4+ T cells exhibit similar recall dynamics, with individual clones giving rise to multiple effectors including highly proliferative Th1/Tr1 cells, as well as GC Tfh and Tfh-like cells lacking proliferative capacity. Thus, we show substantial diversity in recall responses mounted by multiple co-existing CD4+ T cell subsets in the spleen, and present graphical user interfaces for studying gene expression dynamics and clonal relationships during re-infection.
Assuntos
Linfócitos T CD4-Positivos , Malária , Reinfecção , Animais , Malária/imunologia , Malária/parasitologia , Linfócitos T CD4-Positivos/imunologia , Camundongos , Reinfecção/imunologia , Células Th1/imunologia , Camundongos Endogâmicos C57BL , Baço/imunologia , Baço/parasitologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/genética , Camundongos Transgênicos , Feminino , Memória ImunológicaRESUMO
Plasmodium falciparum malaria drives immunoregulatory responses across multiple cell subsets, which protects from immunopathogenesis, but also hampers the development of effective anti-parasitic immunity. Understanding malaria induced tolerogenic responses in specific cell subsets may inform development of strategies to boost protective immunity during drug treatment and vaccination. Here, we analyse the immune landscape with single cell RNA sequencing during P. falciparum malaria. We identify cell type specific responses in sub-clustered major immune cell types. Malaria is associated with an increase in immunosuppressive monocytes, alongside NK and γδ T cells which up-regulate tolerogenic markers. IL-10-producing Tr1 CD4 T cells and IL-10-producing regulatory B cells are also induced. Type I interferon responses are identified across all cell types, suggesting Type I interferon signalling may be linked to induction of immunoregulatory networks during malaria. These findings provide insights into cell-specific and shared immunoregulatory changes during malaria and provide a data resource for further analysis.
Assuntos
Interferon Tipo I , Malária Falciparum , Malária , Humanos , Interleucina-10/genética , Transcriptoma , Interferon Tipo I/genética , Plasmodium falciparum/genética , Subpopulações de Linfócitos TRESUMO
Maturation rates of malaria parasites within red blood cells (RBCs) can be influenced by host nutrient status and circadian rhythm; whether host inflammatory responses can also influence maturation remains less clear. Here, we observed that systemic host inflammation induced in mice by an innate immune stimulus, lipopolysaccharide (LPS), or by ongoing acute Plasmodium infection, slowed the progression of a single cohort of parasites from one generation of RBC to the next. Importantly, plasma from LPS-conditioned or acutely infected mice directly inhibited parasite maturation during in vitro culture, which was not rescued by supplementation, suggesting the emergence of inhibitory factors in plasma. Metabolomic assessments confirmed substantial alterations to the plasma of LPS-conditioned and acutely infected mice, and identified a small number of candidate inhibitory metabolites. Finally, we confirmed rapid parasite responses to systemic host inflammation in vivo using parasite scRNA-seq, noting broad impairment in transcriptional activity and translational capacity specifically in trophozoites but not rings or schizonts. Thus, we provide evidence that systemic host inflammation rapidly triggered transcriptional alterations in circulating blood-stage Plasmodium trophozoites and predict candidate inhibitory metabolites in the plasma that may impair parasite maturation in vivo. IMPORTANCE Malaria parasites cyclically invade, multiply, and burst out of red blood cells. We found that a strong inflammatory response can cause changes to the composition of host plasma, which directly slows down parasite maturation. Thus, our work highlights a new mechanism that limits malaria parasite growth in the bloodstream.
Assuntos
Malária , Parasitos , Camundongos , Animais , Transcriptoma , Lipopolissacarídeos , Malária/parasitologia , Inflamação , Eritrócitos/parasitologiaRESUMO
BACKGROUND: Protective malarial antibodies are acquired more rapidly in adults than children, independently of cumulative exposure, however the cellular responses mediating these differences are unknown. CD4 T-follicular helper (Tfh) cells have key roles in inducing antibodies, with Th2-Tfh cell activation associated with antibody development in malaria. Whether Tfh cell activation in malaria is age dependent is unknown and no studies have compared Tfh cell activation in children and adults with malaria. METHODS: We undertook a comprehensive study of Tfh cells, along with B cells and antibody induction in children and adults with malaria. Activation and proliferation of circulating Tfh (cTfh) cell subsets was measured ex vivo and parasite-specific Tfh cell frequencies and functions studied with Activation Induced Marker (AIM) assays and intracellular cytokine staining. FINDINGS: During acute malaria, the magnitude of cTfh cell activation was higher in adults than in children and occurred across all cTfh cell subsets in adults but was restricted only to the Th1-cTfh subset in children. Further, adults had higher levels of parasite-specific cTfh cells, and cTfh cells which produced more Th2-Tfh associated cytokine IL-4. Consistent with a role of higher Tfh cell activation in rapid immune development in adults, adults had higher activation of B cells during infection and higher induction of antibodies 7 and 28 days after malaria compared to children. INTERPRETATION: Our data provide evidence that age impacts Tfh cell activation during malaria, and that these differences may influence antibody induction after treatment. Findings have important implications for vaccine development in children. FUNDING: This word was supported by the National Health and Medical Research Council of Australia, Wellcome Trust, Charles Darwin University Menzies School of Health Research, Channel 7 Children's Research Foundation, and National Health Institute.
Assuntos
Malária Falciparum , Células T Auxiliares Foliculares , Adulto , Austrália , Linfócitos B , Criança , HumanosRESUMO
Immunity to malaria is mediated by antibodies that block parasite replication to limit parasite burden and prevent disease. Cytophilic antibodies have been consistently shown to be associated with protection, and recent work has improved our understanding of the direct and Fc-mediated mechanisms of protective antibodies. Antibodies also have important roles in vaccine-mediated immunity. Antibody induction is driven by the specialized CD4+ T cells, T-follicular helper (Tfh) cells, which function within the germinal centre to drive B-cell activation and antibody induction. In humans, circulating Tfh cells can be identified in peripheral blood and are differentiated into subsets that appear to have pathogen/vaccination-specific roles in antibody induction. Tfh cell responses are essential for protective immunity from Plasmodium infection in murine models of malaria. Our understanding of the activation of Tfh cells during human malaria infection and the importance of different Tfh cell subsets in antibody development is still emerging. This review will discuss our current knowledge of Tfh cell activation and development in malaria, and the potential avenues and pitfalls of targeting Tfh cells to improve malaria vaccines.
RESUMO
Immunomodulatory therapies can effectively control haematological malignancies. Previously we reported the effectiveness of combination immunotherapies that centre on 4-1BB-targeted co-stimulation of CD8 + T cells, particularly when simultaneously harnessing the immune adjuvant properties of Natural Killer T (NKT) cells. The objective of this study was to assess the effectiveness of agonistic anti-4-1BB antibody-based combination therapy against two aggressive forms of acute myeloid leukemia (AML). Anti-4-1BB treatment alone resulted in transient suppression of established AML-ETO9a tumor growth in 50% of mice, however the majority of these mice subsequently succumbed to disease. Combining alpha-galactosylceramide (α-GalCer)-loaded tumor cell vaccination with anti-4-1BB antibody treatment increased the proportion of responding mice to 100%, and protection led to long-term, tumor-free survival, demonstrating complete eradication of AML. This finding was extended to established mixed lymphocytic leukemia (MLL)-AF9 tumors, whereby vaccine plus anti-4-1BB combination similarly resulted in 100% protection. The addition of anti-PD-1 to anti-4-1BB treatment, although improving survival outcomes compared to anti-4-1BB alone, was not as effective as NKT cell vaccination. The effectiveness of 4-1BB combination therapies was dependent on IFN-γ signaling within host cells, but not tumors. Vaccine plus anti-4-1BB therapy elicited potent generation of functional effector and memory CD8 + T cells in all tumor-associated organs. Therapy induced KLRG1+ effector CD8 T cells were the most effective at controlling disease. We show that combining NKT cell-targeting vaccination with anti-4-1BB provides excellent therapeutic responses against AML and MLL in mice, and these results will guide ongoing efforts in finding immunotherapeutic solutions against acute myeloid leukemias.
RESUMO
A major mechanism of host-mediated control of blood-stage Plasmodium infection is thought to be removal of parasitized red blood cells (pRBCs) from circulation by the spleen or phagocytic system. The rate of parasite removal is thought to be further increased by anti-malarial drug treatment, contributing to the effectiveness of drug therapy. It is difficult to directly compare pRBC removal rates in the presence and absence of treatment, since in the absence of treatment the removal rate of parasites is obscured by the extent of ongoing parasite proliferation. Here, we transfused a single generation of fluorescently-labelled Plasmodium berghei pRBCs into mice, and monitored both their disappearance from circulation, and their replication to produce the next generation of pRBCs. In conjunction with a new mathematical model, we directly estimated host removal of pRBCs during ongoing infection, and after drug treatment. In untreated mice, pRBCs were removed from circulation with a half-life of 15.1â¯h. Treatment with various doses of mefloquine/artesunate did not alter the pRBC removal rate, despite blocking parasite replication effectively. An exception was high dose artesunate, which doubled the rate of pRBC removal (half-life of 9.1â¯h). Phagocyte depletion using clodronate liposomes approximately halved the pRBC removal rate during untreated infection, indicating a role for phagocytes in clearance. We next assessed the importance of pRBC clearance for the decrease in the parasite multiplication rate after high dose artesunate treatment. High dose artesunate decreased parasite replication â¼46-fold compared with saline controls, with inhibition of replication contributing 23-fold of this, and increased pRBC clearance contributing only a further 2.0-fold. Thus, in our in vivo systems, drugs acted primarily by inhibiting parasite replication, with drug-induced increases in pRBC clearance making only minor contributions to overall drug effect.
Assuntos
Antimaláricos/administração & dosagem , Sangue/parasitologia , Malária/tratamento farmacológico , Malária/parasitologia , Carga Parasitária , Parasitemia/parasitologia , Plasmodium berghei/isolamento & purificação , Animais , Artesunato/administração & dosagem , Modelos Animais de Doenças , Fluorescência , Malária/imunologia , Mefloquina/administração & dosagem , Camundongos , Modelos Teóricos , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/isolamento & purificação , Plasmodium berghei/genética , Coloração e RotulagemRESUMO
Combinations of mAbs that target various components of T-cell activation/inhibition may work synergistically to improve antitumor immunity against cancer. In this study, we investigated the therapeutic potential of combining an anticancer vaccination strategy with antibodies targeting an immune stimulatory (4-1BB) and immune inhibitory (PD-1) receptor, in a preclinical model of spontaneously arising c-Myc-driven B-cell lymphoma. In Eµ-myc transgenic mice, we reveal that 4-1BB agonistic mAb treatment alone was sufficient to drive antitumor immunity and prevent disease progression in 70% of mice. When combined with an α-GalCer-loaded, irradiated tumor cell vaccine, 4-1BB mAb treatment led to increased expansion of effector CD8 T-cell populations and protection of long-term surviving mice against tumor rechallenge. Unexpectedly, PD-1 blockade did not provide therapeutic benefit. The T-cell-promoting effects and antitumor activity of 4-1BB mAb were diminished when used simultaneously with a PD-1-blocking mAb. This was associated with a rapid and dramatic reduction in effector CD8+ T-cell subsets in the presence of PD-1 blockade. These findings reveal that supporting T-cell activation therapeutically is effective for controlling B-cell lymphomas; however, caution is required when combining antibody-mediated modulation of both costimulatory and coinhibitory T-cell receptors. Cancer Immunol Res; 5(3); 191-7. ©2017 AACR.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Linfoma de Células B/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Animais , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Modelos Animais de Doenças , Feminino , Galactosilceramidas/administração & dosagem , Memória Imunológica , Imunoterapia , Interferon gama/sangue , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/mortalidade , Linfoma de Células B/patologia , Masculino , Camundongos , Camundongos Transgênicos , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismoRESUMO
The artemisinins are the first-line therapy for severe and uncomplicated malaria, since they cause rapid declines in parasitemia after treatment. Despite this, in vivo mechanisms underlying this rapid decline remain poorly characterised. The overall decline in parasitemia is the net effect of drug inhibition of parasites and host clearance, which competes against any ongoing parasite proliferation. Separating these mechanisms in vivo was not possible through measurements of total parasitemia alone. Therefore, we employed an adoptive transfer approach in which C57BL/6J mice were transfused with Plasmodium berghei ANKA strain-infected, fluorescent red blood cells, and subsequently drug-treated. This approach allowed us to distinguish between the initial drug-treated generation of parasites (Gen0), and their progeny (Gen1). Artesunate efficiently impaired maturation of Gen0 parasites, such that a sufficiently high dose completely arrested maturation after 6h of in vivo exposure. In addition, artesunate-affected parasites were cleared from circulation with a half-life of 6.7h. In vivo cell depletion studies using clodronate liposomes revealed an important role for host phagocytes in the removal of artesunate-affected parasites, particularly ring and trophozoite stages. Finally, we found that a second antimalarial drug, mefloquine, was less effective than artesunate at suppressing parasite maturation and driving host-mediated parasite clearance. Thus, we propose that in vivo artesunate treatment causes rapid decline in parasitemia by arresting parasite maturation and encouraging phagocyte-mediated clearance of parasitised RBCs.